The chemokine family of peptides is defined on the basis of sequence homology and on the presence of variations on a conserved cysteine motif. Schall (1996) Cytokine 3:165-183; and Oppenheim et al. (1991) Annu. Rev. Immunol. 9:617-648. The family can be subdivided on the basis of this motif into two major subfamilies, in which members of each contain four characteristic cysteine residues. This subdivision therefore defines the CC or xcex2-chemokine family in which the first two cysteines are juxtaposed, and the CXC or xcex1-chemokine family in which there is an intervening amino acid between the first two cysteines. Two other subfamilies have subsequently been described which have variations in the number of amino acids between the first two cysteine residues. Kelner et al. (1994) Science 266:1395-1399; Dorner et al. (1997) J. Biol. Chem. 272:8817-8823; and Bazan et al. (1996) Nature 385:640-644.
Chemokines display a range of in vitro and in vivo functions ranging from proinflammatory activities on a range of cell types to proliferative regulatory activities. All of the functions of the chemokine family are believed to be signaled into a responsive cell using members of the G protein-coupled heptahelical receptor family. To date several xcex1-chemokine and xcex2-chemokine receptors have been described. See, for e.g., Neote et al. (1993) Cell 72:415-425; Ponath et al. (1996) J. Exp. Med. 183:2437-2448; and Power et al. (1995) J. Biol. Chem. 270:19495-19500.
The present invention is based, at least in part, on the discovery of novel molecules of the G protein-coupled heptahelical receptor superfamily, referred to herein as xe2x80x9cD6xe2x80x9d nucleic acid and protein molecules. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding D6 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of D6-encoding nucleic acids.
In one embodiment, a D6 nucleic acid molecule is at least about 60-65%, more preferably at least about 70-75%, even more preferably at least about 78-80%, 80-85%, and most preferably at least about 90-95% or more homologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or a complement thereof In yet another embodiment, a D6 nucleic acid molecule is at least about 60-65%, more preferably at least about 70-75%, even more preferably at least about 80-85%, and most preferably at least about 88-95% or more homologous to the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, or a complement thereof. In a preferred embodiment, the isolated nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In another preferred embodiment, the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule comprises nucleotides 1-858 of SEQ ID NO:1 or nucleotides 1-846 of SEQ ID NO:3. In another embodiment, the isolated nucleic acid molecule comprises nucleotides 955-1172 of SEQ ID NO:1 or nucleotides 943-1160 of SEQ ID NO:3. In yet another embodiment, the isolated nucleic acid molecule comprises nucleotides 1-117 or nucleotides 1395-1664 of SEQ ID NO:4.
In another embodiment, a D6 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:5. In another preferred embodiment, a D6 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence 65-70%, preferably at least about, 71-75%, 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ ID NO:2. In another embodiment, a D6 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence 65-70%, preferably at least about, 71-75%, 75-80%, more preferably at least about 85-90%, and most preferably at least about 90-95% or more homologous to the amino acid sequence of SEQ ID NO:5. In yet another embodiment, the nucleic acid molecule encodes the amino acid sequence of human or murine D6.
In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which includes at least two, three, preferably four conserved cysteine residues, and is membrane bound. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which includes at least one G-protein docking motif, at least two conserved cysteine-residues, and is membrane bound. In a further embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which has seven transmembrane domains. In yet another embodiment, a D6 nucleic acid molecule encodes a D6 protein and is a naturally occurring nucleotide sequence.
Another embodiment of the invention features nucleic acid molecules, preferably D6 nucleic acid molecules, which specifically detect D6 nucleic acid molecules relative to nucleic acid molecules encoding non-D6 proteins. For example, in one embodiment, such a nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotides 1-858, 1-69, 955-969, or 955-1172 of the nucleotide sequence shown in SEQ ID NO:1, or to nucleotides 1-846, 1-57, 943-957 or 943-1160 of nucleotide sequence shown in SEQ ID NO:3. In another embodiment, the nucleic acid molecule is at least 215 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, respectively, or a complement thereof. In yet another embodiment, a D6 nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotides 1-117 of the nucleotide sequence shown in SEQ ID NO:4, or to nucleotides 1395-1664 of nucleotide sequence shown in SEQ ID NO:4. In a further embodiment, the nucleic acid molecule is at least 117 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:4 or SEQ ID NO:6, respectively, or a complement thereof.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a D6 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a D6 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing a protein, preferably a D6 protein, by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a D6 protein is produced.
Another aspect of this invention features isolated or recombinant D6 proteins and polypeptides. In one embodiment, an isolated protein, preferably a D6 protein, has at least two, three, preferably four conserved cysteine residues, and is membrane bound. In another embodiment, an isolated protein, preferably a D6 protein, has at least one G-protein docking motif, at least two conserved cysteine-residues, and is membrane bound. In a further embodiment, an isolated protein, preferably a D6 protein, includes seven transmembrane domains. In yet another embodiment, an isolated protein, preferably a D6 protein, has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:5. In a preferred embodiment, a protein, preferably a D6 protein, has an amino acid sequence at least about 65-70%, preferably at least about 71-75%, 75-80%, even more preferably at least about 85-90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, a protein, preferably a D6 protein, has an amino acid sequence at least about 65-70%, preferably at least about 75-80%, even more preferably at least about 85-90%, and most preferably at least about 90-95% or more homologous to the amino acid sequence of SEQ ID NO:5. In another embodiment, a protein, preferably a D6 protein, has the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:5.
In another embodiment, the invention features fragments of the proteins having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, wherein the fragment comprises at least 15 contiguous amino acids of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.
Another embodiment of the invention features an isolated protein, preferably a D6 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 80% homologous to a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. Another embodiment of the invention features an isolated protein, preferably a D6 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 90% homologous to a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complement thereof. This invention further features an isolated protein, preferably a D6 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or SEQ ID NO:3, or a complement thereof. In another embodiment, an isolated protein, preferably a D6 protein, is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4, or SEQ ID NO:6, or a complement thereof.
The proteins of the present invention, preferably D6 proteins, or biologically active portions thereof, can be operatively linked to a non-D6 polypeptide to form fusion proteins. The invention further features antibodies that specifically bind proteins, preferably D6 proteins, such as monoclonal or polyclonal antibodies. In addition, the D6 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting D6 expression in a biological sample by contacting the biological sample with an agent capable of detecting a D6 nucleic acid molecule, protein or polypeptide such that the presence of a D6 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of D6 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of D6 activity such that the presence of D6 activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating D6 activity comprising contacting the cell with an agent that modulates D6 activity such that D6 activity in the cell is modulated. In one embodiment, the agent inhibits D6 activity. In another embodiment, the agent stimulates D6 activity. In one embodiment, the agent is an antibody that specifically binds to a D6 protein. In another embodiment, the agent modulates expression of D6 by modulating transcription of a D6 gene or translation of a D6 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a D6 mRNA or a D6 gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant D6 protein or nucleic acid expression or activity by administering an agent which is a D6 modulator to the subject. Another embodiment of the invention features a method of treating a subject having a disorder characterized by aberrant chemokine expression or activity by administering an agent which is a D6 modulator to the subject. In one embodiment, the D6 modulator is a D6 protein. In another embodiment, the D6 modulator is a D6 nucleic acid molecule. In yet another embodiment, the D6 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant D6 protein or nucleic acid expression or aberrant chemokine expression is an inflammatory or proliferative disorder.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a D6 protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of a D6 protein, wherein a wild-type form of said gene encodes an protein with a D6 activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of a D6 protein, by providing an indicator composition comprising a D6 protein having D6 activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on D6 activity in the indicator composition to identify a compound that modulates the activity of a D6 protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.